The detection and quantification of specific biomolecules, ions, or metabolites are important for in vivo real-time monitoring of cellular processes and for in vitro biosensing and clinical diagnosis. Of the fluorescent probes used for these applications, those that enable detection without separation from the target are more desirable, especially for intracellular studies where removal of unbound probes is difficult. To this end, a number of detection strategies have been developed using reporters that fluoresce upon probe-target binding, including split green fluorescent proteins, electron transfer-based probes, biarsenic organic dyes intercalating dyes, fluorescence resonance energy transfer (FRET)-based indicators, and molecular beacons.
A molecular beacon (“MB”) is hairpin-shaped nucleic acid probe that fluoresces upon hybridization with a specific DNA target. MBs have been used much since their introduction in 1996. While successful as a separation-free probe, a MB is generally limited by background fluorescence that comes from imperfect quenching of donors and conformational fluctuations of the hairpin structure. Well-designed MBs can achieve a signal-to-background ratio (S/B ratio) of more than 100-fold but often require special quenchers or sophisticated thermodynamic analysis for stem-loop sequence selection. Additionally, a MB needs to be labeled with a donor fluorophore and a quencher. Therefore, MBs suffer from problems associated with double labeling (e.g. high cost, low yield, singly labeled impurities, extensive purification etc).
Better probes and methods for detecting a target DNA are desirable.
Noble metal nanoclusters, such as those made of silver, gold, copper or other noble metals are collections of small numbers of metal atoms (2-30 atoms) with physical sizes close to the Fermi wavelength of an electron (˜0.5 nm for gold and silver). They behave like molecular systems and yield fluorescence emission in the UV-visible range. For example, it has been known for a few years that certain DNA bases and sequences can act as templates for stabilization of fluorescent silver nanoclusters. The resulting oligonucleotide-templated silver nanoclusters (“DNA/Ag NCs”) are a versatile set of fluorophores. They have been used for a variety of applications including live cell imaging, detection of specific metal ions, and single-nucleotide variation identification. These DNA/Ag NCs are very small, relatively simple to prepare, and biocompatible (they are made of Ag). They have much better photostability than commonly used organic dyes and may also be a few times brighter. Unlike organic dyes and photoluminescent nanocrystals, they are subject to silver oxidation/reduction or nanocluster (“NC”) regrouping, which results in conversion among different NC species. These different species may have different colors. The conversion amongst different NC species is not well understood, but may be reversible and depends on a number of factors including time, temperature, oxygen and salt content.
An object of the invention is to provide a probe for detecting a target DNA by fluorescence emission upon hybridization with the target DNA.
Another object of the invention is to provide a method for detecting a target DNA using a probe that produces an enhanced fluorescence emission upon hybridizing with the target DNA.